Characterization of high molecular weight glutenin subunit genes from the Ns genome of Psathyrostachys juncea

The Ns genome of the genus Psathyrostachys possesses superior traits useful for wheat improvement. However, very little is known about the high molecular weight (HMW) subunits of glutenin encoded by the Ns genome. In this paper, we report the isolation of four alleles of HMW glutenin subunit gene from Psathyrostachys juncea. Sequence alignment data shows the four alleles have similar primary structure with those in wheat and other wheat-related grasses, with some unique modifications. All four sequences more closely resemble y-type, rather than x-type, glutenins. However, our results show three of the subunits (1Ns2-4) contain an extra glutamine residue in the N-terminal region not found on typical y-type subunits, as well as the x-type subunit specific sequence LAAQLPAMCRL. These three subunits likely represent an intermediate state in the divergence between x- and y-type subunits. Results also indicate that the Ns genome is more closely related to the St genome of Pseudoroegneria than any other Triticeae genomes.     

Molecular characterization of LMW-GS genes from a somatic hybrid introgression line Ⅱ-12 between Triticum aestivum and Agropyron elongatum in relation to quick evolution

In order to exploit the evolution and find novel low-molecular-weight glutenin subunit(LMW-GS)for improvement of common wheat quality,thirteen variants from a somatic hybrid introgression line Ⅱ-12 between Triticum aestivum cv.Jinan 177(JN177)and Agropyron elongatum were characterized via genomic PCR.Four clones were pseudogenes because they contained an internal stop codon.The remaining nine variants contained intact open reading frames(ORFs).Sequence alignment indicates that the proteins deduced from the nine ORFs have similar primary structure with LMW-GS cloned from its parents previously.However,they have some unique modifications in the structures.For example,EU292737 contains not only an extra Cys residue in the C-terminal domain but also a long repetitive domain.Both EU159511 and EU292738 start their first Cys residue in the N-terminal repetitive domain,but not in the N-conserved domain traditionally.These structural alterations may have positive contributions to wheat flour quality.The results of phylogeny showed that most LMW-GS variances from Ⅱ-12 were homologous to those from parent JN 177 and other wheat lines.The reason for quick evolution of LMW-GS in Ⅱ-12 was discussed.     

高冰草中一种新型高分子量麦谷蛋白亚基编码序列的研究

高冰草(Agropyron elongatun)是普通小麦(Triticum aestivum)的近缘禾草,SDS-PAGE显示其所编码的麦谷蛋白亚基的类型较普通小麦更加丰富,是普通小麦品质改良的重要亲本之一。利用基因组PCR的方法从高冰草中克隆到一个新的高分子量麦谷蛋白亚基(HMW-GS)基因(AgeloG2)全编码序列,同源性分析表明：与普通小麦的1Dy12基因比较在少数位点发生了碱基替换和一处6碱基序列的缺失,同源性为99％;与普通小麦的1Dy10基因比较,该基因亦只有少数碱基的替换和两处18碱基序列的增加及一处6碱基序列的缺失,同源性为98％。从推导的编码序列分析,AgeloG2编码y型HMW—GS。综上分析,AgeloG2是一个新的高分子量麦谷蛋白y-型亚基基因。聚类分析结果显示,无论在基因序列还是推导的氨基酸序列上,小麦1Dy亚基与AgeloG2的同源性都高于与粗山羊来源的y型亚基的同源性。     

LMW-GS genes in Agropyron elongatum and their potential value in wheat breeding

To study the usefulness of low-molecular-weight glutenin subunits (LMW-GS) of Agropyron elongatum (Host) Nevski to wheat (Triticum aestivum L.) quality improvement, we characterized LMW-GS genes of A. elongatum. Nine LMW-GS genes of A. elongatum, which were named AeL1 to AeL9, were cloned by genomic PCR. After sequencing, we obtained complete open reading frames from AeL2 to AeL8 and partial genes of AeL1 and AeL9. All nine sequences are homoeologous to those of wheat and related grasses. Comparison of the deduced amino acid sequences with those of published LMW-GS suggests that the basic structures of all the subunits are very similar. However, except for AeL4 and AeL5, which contain the identical N-terminal sequence with LMW-m, other LMW-GS sequences separated from A. elongatum cannot be classified according to previous criteria for the three types: LMW-m (methionine), LMW-s (serine), and LMW-i (isoleucine), and then 12 groups. In addition, there are some characters in the LMW-GS sequences of A. elongatum: AeL2, AeL3, and AeL6 involve a Cys residue in the signal peptide respectively, which is absent in most of LMW-GS; AeL3, AeL6, AeL8, and AeL9 start their first Cys residues in the N-terminal repetitive domains, respectively; both AeL2 and AeL5 have nine Cys residues, with an extra Cys residue in the N-terminal repetitive domain and the repetitive and glutamine-rich domain; AeL2, AeL3, AeL6, and AeL9 comprise long repetitive domains. Phylogenetic analysis indicates that there is a relatively weak sequence identity between the LMW-GS genes from A. elongatum cloned in this study and those reported from other plants. Three LMW-GS sequences, AeL2, AeL3, and AeL6, are clustered to Glu-A3 from wheat than to those from other plants. The possible use of these genes in relation to the high quality of hybrid wheat is discussed.     

Characterizing HMW-GS alleles of decaploid <Emphasis Type="Italic">Agropyron elongatum</Emphasis> in relation to evolution and wheat breeding

Bread wheat quality is mainly correlated with high molecular weight glutenin subunits (HMW-GS) of endosperm. The number of HMW-GS alleles with good processing quality is limited in bread wheat cultivars, while there are plenty of HMW-GS alleles in wheat-related grasses to exploit. We report here on the cloning and characterization of HMW-GS alleles from the decaploid Agropyron elongatum. Eleven novel HMW-GS alleles were cloned from the grass. Of them, five are x-type and six y-type glutenin subunit genes. Three alleles Aex4, Aey7, and Aey9 showed high similarity with another three alleles from the diploid Lophopyrum elongatum, which provided direct evidence for the E^e genome origination of A. elongatum. It was noted that C-terminal regions of three alleles of the y-type genes Aey8, Aey9, and Aey10 showed more similarity with x-type genes than with other y-type genes. This demonstrates that there is a kind of intermediate state that appeared in the divergence between x- and y-type genes in the HMW-GS evolution. One x-type subunit, Aex4, with an additional cysteine residue, was speculated to be correlated with the good processing quality of wheat introgression lines. Aey4 was deduced to be a chimeric gene from the recombination between another two genes. How the HMW-GS genes of A. elongatum may contribute to the improvement of wheat processing quality are discussed.     

用RAPD技术鉴定小麦属间不对称体细胞杂种

用随机引物扩增多态DNA(RAPD)技术对三种不同组合:小麦(Triticum aestivum)( )簇毛麦(Haynaldia villosa);小麦( )羊草(Leymus chinensis)和小麦( )高冰草(Agropyron elongatum)的属间不对称杂种进行分子鉴定,不同杂种植株的基因组经随机引物扩增后,均出现双亲的多态特异产物,证实它们含有双亲的基因组。将引物OPJ-12扩增的高冰草多态特异产物(分子量为0.77bp的DNA片段)分离纯化并标记作探针,用Southern杂交证明了小麦( )高冰草杂种经OPJ-12扩增的0.77kbp特异片段与高冰草这一片段具有同源性。本文结果证明,RAPD技术可作为小麦属间不对称体细胞杂种的一种快速、简便、有效的分子鉴定方法。     

RAPD method for the identification of intergeneric asymmetric somatic hybrid plants of wheat]

Randomly amplified polymorphic DNA (RAPD) method was used to identify the hybrid nature of three kinds of intergeneric asymmetric somatic hybrid plants of wheat: wheat (Triticum aestivum) + Haynaldia villosa, Wheat + Leymus chinensis and wheat + Agropyron elongatum. It was shown from the electrophoresis profiles that the genome of somatic hybrid plants contained specific section genome of both parents after DNA amplification with arbitrary primers. A specific RAPD product (DNA fragment of 0.77 kbp) of A. elongatum generated with primer OPJ-12 was isolated, purified, labeled and used as a probe. Southern blot from OPJ-12 primer-generated specific section genome of the hybrid (T. aestivum + A. elongatum) hybridized to this probe (0.77 kbp) proved that they are homologous in nature. This paper also discussed the advantage of RAPD method in identification of hybrid plants, especially asymmetric somatic hybrids.     

小麦与高冰草体细胞杂种后代的核基因组和叶绿体基因组的遗传特征

以小麦(Triticum aestivum L.)与高冰草(Agropyron elongatum(Host)Nevski)体细胞杂种同一个克隆来源的F2-F6自交系Ⅱ-2、Ⅱ-Ⅰ-8以及由Ⅱ-Ⅰ-8 F2分离形成的8-1(F3-F6)为材料,利用小麦叶绿体基因组的微卫星(Microsatellite)特异引物及随机扩增多态性DNA(RAPD)引物进行分析.结果表明,杂种株系的叶绿体基因组组成一致,均以小麦叶绿体基因组为主,仅在rpl14和rpl16基因的间隔序列中检测到双亲的特征带,表明有高冰草的叶绿体DNA在杂种中存在,并稳定遗传至第六代.RAPD分析表明,不同杂种株系中存在不同的高冰草核DNA片段,核基因组在传代中基本稳定.     

应用基因组原位杂交鉴定蓝粒小麦及其诱变后代

应用基因组原位杂交技术（GISH）对普通小麦（Triticum aestivumL.)和长穗偃麦草[Agropyron elongatum(Host)Beauv,2n=10x=70]杂交后选育出的蓝粒小麦蓝-58及其诱变后代的染色体组成进行了鉴定。结果表明,GISH可方便地检测到小麦遗传背景中的长穗偃麦草染色体或易位的片段。如前人报道,蓝-58（2n=42)是一个具有2条长穗偃麦草4E染色体的异代换系（4E/4D）。LW004可能是一个具有两对相互易位染色体的纯合系,其田间表现磷高效特性,LW43-3-4为41条染色体的蓝单体（40W 1’4E）,种子颜色为浅蓝色,通过此法还检测出一些染色体结构发生很大变异的材料如4E的单端体（40W 1‘4E),种子颜色为浅蓝色,通过此法还检测出一些染色结构发生很大变异的材料如4E的单端体（40W 1‘t4E)以及组型为39W 1‘4E 1‘t4E的个体,此项研究结果更为直观地表明控制蓝粒体状的基因的确在来自长穗偃麦草的染色体上。同时说明有效的突变方法与灵活方便的检测手段的有机结合在染色体工程材料的创制和染色体工程育种中起着至关重要的作用。     

长穗偃麦草优异基因的染色体定位及应用

长穗偃麦草比较公认的有2个种,即二倍体长穗偃麦草(Thinopyrum elongatum,2n=2X)和十倍体长穗偃麦草(Thinopyrum ponticum,2n=10X),是重要的小麦近缘种,具有抗病、抗寒、抗旱、耐盐碱等优良性状。因其基因组中蕴含许多对小麦品种改良极为有用的基因,且易与小麦杂交等优势,多年来长穗偃麦草一直作为小麦遗传改良的优良种质资源而备受关注。本文对长穗偃麦草的基因组研究及其在小麦的抗逆、抗病和提高光合能力、产量及高分子量谷蛋白(HMW-GS)含量等方面的应用做了综述,为其基因组中优异基因的进一步开发和利用提供了理论依据。     

Molecular cytogenetic characterization of wheat--Thinopyrum elongatum addition, substitution and translocation lines with a novel source of resistance to wheat Fusarium Head Blight

Thinopyrum elongatum(2n = 2x = 14,EE),a wild relative of wheat,has been suggested as a potentially novel source of resistance to several major wheat diseases including Fusarium Head Blight(FHB).In this study,a series of wheat(cv.Chinese Spring,CS) substitution and ditelosomic lines,including Th.elongatum additions,were assessed for TypeⅡresistance to FHB.Results indicated that the lines containing chromosome 7E of Th.elongatum gave a high level of resistance to FHB,wherein the infection did not spread beyond the inoculated floret.Furthermore,it was determined that the novel resistance gene(s) of 7E was located on the short-arm(7ES) based on sharp difference in FHB resistance between the two 7E ditelosomic lines for each arm.On the other hand,Th.elongatum chromosomes 5E and 6E likely contain gene(s) for susceptibility to FHB because the disease spreads rapidly within the inoculated spikes of these lines. Genomic in situ hybridization(GISH) analysis revealed that the alien chromosomes in the addition and substitution lines were intact,and the lines did not contain discernible genomic aberrations.GISH and multicolor-GISH analyses were further performed on three translocation lines that also showed high levels of resistance to FHB.Lines TA3499 and TA3695 were shown to contain one pair of wheat-Th. elongatum translocated chromosomes involving fragments of 7D plus a segment of the 7E,while line TA3493 was found to contain one pair of wheat-Th.elongatum translocated chromosomes involving the D- and A-genome chromosomes of wheat.Thus,this study has established that the short-arm of chromosome 7E of Th.elongatum harbors gene(s) highly resistant to the spreading of FHB,and chromatin of 7E introgressed into wheat chromosomes largely retained the resistance,implicating the feasibility of using these lines as novel material for breeding FHB-resistant wheat cultivars.     

RAPD and ISSR-assisted identification and development of three new SCAR markers specific for the Thinopyrum elongatum E (Poaceae) genome

Diploid Thinopyrum elongatum, a wild relative of wheat, contains many agronomically desirable traits and has potential for increasing genetic variability and introducing desirable characters in this crop. Few molecular markers are available for rapid screening of T. elongatum genome segments in the wheat genetic background. We used 36 RAPD primers and 33 ISSR primers to screen for polymorphisms in the common wheat variety Chinese Spring and in T. elongatum. Two RAPD markers and one ISSR marker, designated OPF03(1407), LW10(1487) and UBC841(701), were identified and were specific for the T. elongatum E genome. Three pairs of primers flanking these specific sequences were designed to produce SCAR markers. All three SCAR markers were T. elongatum E genome-specific. Two of these SCAR markers, SCAR(807) and SCAR(577), were present in all seven T. elongatum chromosomes, while SCAR(839) was specific for T. elongatum chromosomes 2E and 3E. These newly developed SCAR markers should be useful for detecting alien genome chromatin or chromosome segments in the genetic background of common wheat.     

EST-derived SSR markers from defined regions of the wheat genome to identify Lophopyrum elongatum specific loci.

Lophopyrum elongatum, a close relative of wheat, provides a source of novel genes for wheat improvement. Molecular markers were developed to monitor the introgression of L. elongatum chromosome segments into hexaploid wheat. Existing simple sequence repeats (SSRs) derived from genomic libraries were initially screened for detecting L. elongatum loci in wheat, but only 6 of the 163 markers tested were successful. To increase detection of L. elongatum specific loci, 165 SSRs were identified from wheat expressed sequence tags (ESTs), where their chromosomal positions in wheat were known from deletion bin mapping. Detailed sequence analysis identified 41 SSRs within this group as potentially superior in their ability to detect L. elongatum loci. BLASTN alignments were used to position primers within regions of the ESTs that have sequence conservation with at least 1 similar EST from another cereal species. The targeting of primers in this manner enabled 14 L. elongatum markers from 41 wheat ESTs to be identified, whereas only 2 from 124 primers designed in random positions flanking SSRs detected L. elongatum loci. Addition and ditelosomic lines were used to assign all 22 markers to specific chromosome locations in L. elongatum. Nine of these SSR markers were assigned to homoeologous chromosome locations based on their similar position in hexaploid wheat. The remaining markers mapped to other L. elongatum chromosomes indicating a degree of chromosome rearrangements, paralogous sequences and (or) sequence variation between the 2 species. The EST-SSR markers were also used to screen other wheatgrass species indicating further chromosome rearrangements and (or) sequence variation between wheatgrass genomes. This study details methodologies for the generation of SSRs for detecting L. elongatum loci.     

应用荧光原位杂交和染色体配对研究八倍体小冰麦中2的染色体组构成及染色体特征

通过染色体配对分析和荧光原位杂交（ＦＩＳＨ）技术对八倍体小冰麦中２的染色体组构成进行分析,结果表明：八倍体小冰麦中２含有的冰草染色体是来自天蓝冰草（Ａｇｒｏｐｙｒｏｎ　ｉｎｔｅｒｍｅｄｉｕｍ（Ｈｏｓｔ）Ｐ．Ｂ．＝Ｅｌｙｔｒｉｇｉａ　ｉｎｔｅｒｍｅｄｉａ（Ｈｏｓｔ）Ｎｅｖｓｋｉ＝Ｔｈｉｎｏｐｙｒｕｍ　ｉｎｔｅｒｍｅｄｉｕｍ　（Ｈｏｓｔ）Ｂａｒｋｗｏｒｔｈ　ａｎｄ　Ｄｅｗｅｙ）具同亲关系的染色体组,但冰草的这种同亲关系的染色体组不同于二倍体长穗偃麦草（Ｔｈｉｎｏｐｙｒｕｍ　ｅｌｏｕｇａｔｕｍ　２Ｘ）的Ｅ组染色体。中２含有１２条冰草染色体,且有一对染色体为小麦（Ｔｒｉｔｉｃｕｍ　ａｅｓｔｉｖｕｍ　Ｌ．）染色体和冰草染色体之间易位所形成的。     

Whole-genome sequencing uncovers the structural and transcriptomic landscape of hexaploid wheat/Ambylopyrum muticum introgression lines

Wheat is a globally vital crop, but its limited genetic variation creates a challenge for breeders aiming to maintain or accelerate agricultural improvements over time. Introducing novel genes and alleles from wheat's wild relatives into the wheat breeding pool via introgression lines is an important component of overcoming this low variation but is constrained by poor genomic resolution and limited understanding of the genomic impact of introgression breeding programmes. By sequencing 17 hexaploid wheat/Ambylopyrum muticum introgression lines and the parent lines, we have precisely pinpointed the borders of introgressed segments, most of which occur within genes. We report a genome assembly and annotation of Am. muticum that has facilitated the identification of Am. muticum resistance genes commonly introgressed in lines resistant to stripe rust. Our analysis has identified an abundance of structural disruption and homoeologous pairing across the introgression lines, likely caused by the suppressed Ph1 locus. mRNAseq analysis of six of these introgression lines revealed that novel introgressed genes are rarely expressed and those that directly replace a wheat orthologue have a tendency towards downregulation, with no discernible compensation in the expression of homoeologous copies. This study explores the genomic impact of introgression breeding and provides a schematic that can be followed to characterize introgression lines and identify segments and candidate genes underlying the phenotype. This will facilitate more effective utilization of introgression pre-breeding material in wheat breeding programmes.     

Identification of the parental chromosomes of the wheat-alien amphiploid Agrotana by genomic in situ hybridization.

Labelled total genomic DNA from four alien species, Thinopyrum ponticum (Host) Beauv. (2n = 70, genomes J1J1J1J2J2), Th. bessarabicum (Savul. &Rayss) Love (2n = 14, genome J), Th. elongatum (Host) Beauv. (2n = 14, genome E), and Haynaldia villosa (L.) Schur. (2n = 14, genome V), were used as probes in combination with blocking wheat DNA for in situ hybridization of the chromosomes of Agrotana, a wheat-alien hybrid (2n = 56) of unknown origin. The results showed that genomic DNA probes from Th. ponticum and Th. bessarabicum both clearly revealed 16 alien and 40 wheat chromosomes in Agrotana, indicating that the J genome present in these two species has a high degree of homology with the alien chromosomes in Agrotana. Biotinylated genomic DNA probe from Th. elongatum identified 10 chromosomes from Agrotana, while some regions of six other chromosomes yielded a weak or no signal. The probe from H. villosa produced no differential labelling of the chromosomes of Agrotana. The genomic formula of Agrotana was designated as AABBDDJJ. We suggest that the alien parent donor species of Agrotana is Th. ponticum rather than Th. bessarabicum. Genomic relationships of the three Thinopyrum species are discussed in relation to the distribution of GISH signals in the chromosomes of Agrotana.     

Wheat genetic diversity trends during domestication and breeding

It has been claimed that plant breeding reduces genetic diversity in elite germplasm which could seriously jeopardize the continued ability to improve crops. The main objective of this study was to examine the loss of genetic diversity in spring bread wheat during (1) its domestication, (2) the change from traditional landrace cultivars (LCs) to modern breeding varieties, and (3) 50 years of international breeding. We studied 253 CIMMYT or CIMMYT-related modern wheat cultivars, LCs, and Triticum tauschii accessions, the D-genome donor of wheat, with 90 simple sequence repeat (SSR) markers dispersed across the wheat genome. A loss of genetic diversity was observed from T. tauschii to the LCs, and from the LCs to the elite breeding germplasm. Wheats genetic diversity was narrowed from 1950 to 1989, but was enhanced from 1990 to 1997. Our results indicate that breeders averted the narrowing of the wheat germplasm base and subsequently increased the genetic diversity through the introgression of novel materials. The LCs and T. tauschii contain numerous unique alleles that were absent in modern spring bread wheat cultivars. Consequently, both the LCs and T. tauschii represent useful sources for broadening the genetic base of elite wheat breeding germplasm.     

部分耐盐小麦品种(系)SSR位点遗传多样性研究

选择有多态性的32对SSR引物对80个小麦耐盐品种(系)进行遗传差异研究,共检测出155个等位变异,平均每个位点上有4.75个等位变异;供试80份耐盐小麦品种(系)来源广泛,遗传基础丰富,表现出较高的遗传多样性,遗传相似系数范围在0.26～0.81;聚类分析结果显示,冬性小麦品种(系)聚为一大类;春性小麦品种(系)也聚为一大类;一些系谱相同或相近的品种(系)遗传相似系数较大;A、B、D基因组中SSR位点平均等位变异差异不大,以B基因组较高.